Project Summary The long term goal of this project is to establish an efficient system for the culture of human spermatogonial stem cells (SSCs). Availability of safe and efficient strategies to propagate SSCs in culture will aid in the development of new options for male factor infertility as well as preservation and restoration of fertility in juvenile and adult patients undergoing potentially sterilizing treatments for cancer. This project will use pigs as an established, accessible animal model phylogenetically and physiologically more similar to humans than rodents. Key findings will be translated to human SSCs. Lack of appropriate tools to monitor SSC function, to generate sufficient numbers of SSCs for fertility preservation, and to develop alternatives to transplantation for screening of SSC function are identified as significant barriers for translation to new treatment options for male infertility (PAR 16-114). Three specific aims address these barriers: 1) To characterize autophagy as a functional biomarker in SSCs, 2) To investigate stirred suspension bioreactors as a novel approach to SSC culture, and 3) To explore testicular organoid formation as a short term functional assay for SSCs. Aim 1 will explore activation of the autophagic pathway in SSCs in response to stressors associated with removal from the testis microenvironment, culture in vitro and exposure to toxic insults. This is expected to establish autophagy as a functional biomarker to improve culture conditions and as a toxicological bioassay for germ line stem cells. Aim 2 will focus on establishing culture in stirred suspension bioreactors as an original scalable approach to maintain and expand SSCs in vitro. Our team members are pioneers of this approach, having established its use for culture of mouse and human pluripotent stem cells in an undifferentiated state, and it has shown early promise for SSC culture. Experiments also address strategies to isolate undifferentiated spermatogonia from the donor testis while minimizing cell stress, thus initiating a suitable cell population for culture. As homologous transplantation is not a viable bioassay for stem cell potential in non-rodent SSCs, support of spermatogenesis in de novo formed testis tissue after grafting to mouse host, as previously validated by our team, will serve as a functional endpoint. Aim 3 will explore testicular organoid formation in microwell culture as an alternative bioassay for transplantation. We will draw on expertise of our collaborator who invented culture of functional organoids in microwells. Formation of organoids with appropriate cell associations will be monitored by confocal microscopy and correlated with support of spermatogenesis after grafting in vivo. If parameters identified in microwell culture are predictive of the ability of SSCs to support sperm production after transplantation, microwell organoid culture could serve as a functional short-term screening assay for SSCs. Taken together, our experimental results will provide an integrated system to isolate, culture and functionally analyze non-rodent SSCs with clinical translation towards the preservation and restoration of human fertility.